Selective near-infrared optical imaging of necrotic cells and simultaneous cell fixing and counter staining with metallacrown complexes

ABSTRACT

In an example of a method for simultaneously fixing and staining cells, the cells are initially incubated in a solution including a Ln(III)Zn 16 (HA ligand) 16  metallacrown complex, wherein the HA ligand is a hydroximate ligand. The incubating cells are exposed to ultraviolet (UV) light. The cells are allowed to continue to incubate in the solution after UV light exposure.

BACKGROUND

Optical devices, bioanalytical assays, and biological imaging probesoften utilize components that exhibit optical properties, such asorganic fluorophores and semi-conductor nanoparticles. Some desiredoptical properties include long luminescence lifetimes, large effectiveenergy differences between excitation and emission bands, and sharpemission bands throughout the visible and near-infrared (NIR) spectralranges. NIR optical imaging has clinical potential to significantlyimprove diagnosis of various human diseases in real time imagingexperiments. Currently used nucleic acid-binding NIR dyes (e.g., organicfluorophores) are not fully reliable for optical imaging, in partbecause of low quantum yield, broad bandwith, high energy excitationwavelengths, small Stokes shift, poor water solubility, and lowphotostability (i.e., prone to photobleaching). These characteristics ofnucleic acid-binding NIR dyes can limit detection sensitivity anddeleteriously affect image resolution. While quantum dots have betterresistance to photobleaching and higher quantum yields than nucleicacid-binding NIR dyes, these imaging agents may suffer from blinking(i.e., random fluctuations in light emission) and may be toxic for invivo applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of examples of the present disclosure will become apparent byreference to the following detailed description and drawings, in whichlike reference numerals correspond to similar, though perhaps notidentical, components. For the sake of brevity, reference numerals orfeatures having a previously described function may or may not bedescribed in connection with other drawings in which they appear.

FIG. 1 is a graph illustrating the normalized emission spectra oflanthanides;

FIG. 2 illustrates an x-ray crystallographic representation ofYb³⁺[Zn(II)MC_(pyzHA)] complex, and the pyrazinehydroxamic acid(H₂pyzHA) from which it was formed;

FIG. 3. illustrates the absorption spectra of H₂pyzHA (multiplied by afactor of 16), Yb³⁺[Zn(II)MCpyzHA] and Nd³⁺[Zn(II)MCpyzHA] MCs in water(150 μM, room temperature).

FIG. 4. illustrates the normalized excitation (λ_(em)(Nd³⁺)=1070 nm,λ_(em)(Yb³⁺)=980 nm) and emission (λ_(ex)=370 nm) spectra ofYb³⁺[Zn(II)MC_(pyzHA)], at the top, and Nd³⁺[Zn(II)MC_(pyzHA)] MCs, atthe bottom, (solid or 200 μM solutions, room temperature).

FIGS. 5A and 5B are graphs illustrating the photostability of theYb³⁺[Zn(II)MC_(pyzHA)], the Nd³⁺[Zn(II)MC_(pyzHA)], and/or propidiumiodide in different media;

FIG. 6 is a graph depicting the results of a cytotoxicity test of HeLacells incubated in with Yb³⁺[Zn(II)MC_(pyzHA)] for 24 an 48 hours, andthen in Alamar blue;

FIGS. 7A through 7E are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 45 μM ofYb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, washed with OPTI-MEM® media andincubated with 3 μM propidium iodide for 5 minutes. FIG. 7A is thebrightfield image; FIG. 7B is the image of the emission signal arisingfrom Yb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (λ_(em)=805 nm long passfilter) obtained after 10 s of exposure to excitation (λ_(ex)=447 nm,filter with 60 nm bandwidth); FIG. 7C shows visible signal arising frompropidium iodide obtained after 100 ms of exposure time (λ_(em)=605 nm,band pass filter with 70 nm bandwidth; and λ_(ex)=550 nm, band passfilter with 25 nm bandwidth); FIG. 7D shows the merging of FIGS. 7B and7C; and FIG. 7E shows the merging of FIGS. 7A, 7B, and 7C;

FIGS. 8A to 8J illustrate results of photobleaching experiments obtainedby epifluorescence microscopy in necrotic HeLa cells incubated with 45μM Yb³⁺[Zn(II)MC_(pyzHA)] for 15 min or 3 μM PI for 5 min. (Top) PI,visible emission (λ_(ex)=550 nm, band pass filter with 25 nm bandwidth;λ_(em)=605 nm, band pass filter with 70 nm bandwidth; exposure time: 100ms) after a continuous excitation with a 550 nm band pass filter (25 nmbandwidth) during: (8A) 10 s, (8B) 50 s, (8C) 100 s, (8D) 200 s, (8E)500 s. (Bottom) Yb³⁺[Zn(II)MC_(pyzHA)], NIR emission arising from Yb³⁺(λ_(ex)=447 nm, band pass filter with 60 nm bandwidth; λ_(em)=805 nm,longpass filter; exposure time: 10 s) after continuous excitation with a447 nm band pass filter (60 nm bandwidth) during: (8F) 10 s, (8G) 50 s,(8H) 100 s, (8I) 200 s, (8J) 500 s. 63× objective.

FIGS. 9A through 9E are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 150 μM ofYb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, illuminated with UV light (377 nmband pass filter with 50 nm bandwidth) for 8 minutes, further incubatedfor 1 hour, washed with OPTI-MEM® media and incubated with 3 μMpropidium iodide for 5 minutes. FIG. 9A is the brightfield image; FIG.9B is the image of the emission signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (λ_(em)=805 nm, longpass filter)obtained after 8 s of exposure to excitation (λ_(ex)=447 nm, band passfilter with 60 nm bandwidth); FIG. 9C shows the image of the visiblesignal arising from propidium iodide obtained after 800 ms of exposuretime (λ_(em)=605 nm, band pass filter with 70 nm bandwidth; andλ_(ex)=550 nm, band pass filter with 25 nm bandwidth); FIG. 9D is FIGS.9B and 9C merged together; and FIG. 9E is FIGS. 9A, 9B, and 9C mergedtogether;

FIGS. 10A through 10C are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 150 μMNd³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, illuminated with UV light (377nm) for 5 minutes, further incubated for 1 hour, washed with OPTI-MEM®media. FIG. 10A is the brightfield image; FIG. 10B is the image of theemission signal arising from Nd³⁺[Zn(II)MC_(pyzHA)] in the NIR range(λ_(em)=805 nm, longpass filter) obtained after 12 s of exposure toexcitation (λ_(ex)=377 nm, band pass filter with 50 nm bandwidth); andFIG. 10C illustrates the merging of FIGS. 10A and 10B;

FIGS. 11A and 11B illustrate results of epifluorescence microscopyexperiments performed on HeLa cells fixed with Yb³⁺[Zn(II)MC_(pyzHA)].Detection of the NIR emission with a standard CCD camera (Orca-R2,Hamamatsu): (A) λ_(ex): 447 nm band pass filter with 60 nm bandwidth,λ_(em): 805 nm longpass filter, exposure time: 30 s, (B) λ_(ex): 447 nmband pass filter with 60 nm bandwidth, λ_(em): 996 nm band pass filterwith 70 nm bandwidth, exposure time: 80 s. 63× objective.

FIGS. 12A to 12E illustrate results of epifluorescence microscopyexperiments on HeLa cells fixed with 150 μM Yb³⁺[Zn(II)MC_(pyzHA)] andillumination with the UV-A light obtained through a 377 nm band passfilter (50 nm bandwidth) for 5 min After fixation, the cells were washedand incubated with 3 μM PI for 5 min. (12A) Brightfield. (12B) NIRsignal arising from Yb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band passfilter with 60 nm bandwidth, λ_(em): 805 nm longpass filter, exposuretime: 8 s). (12C) Visible signal arising from PI (λ_(ex): 550 nm bandpass filter with 25 nm bandwidth, λ_(em): 605 nm band pass filter with70 nm bandwidth, exposure time: 800 ms). (12D) Merged image obtained bythe combination of the PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (12E)Merged image obtained by the combination of the PI,Yb³⁺[Zn(II)MC_(pyzHA)] and brightfield images. 63× objective.

FIGS. 13A to 13E illustrate results of epifluorescence microscopyexperiments on HeLa cells fixed with 150 uM Yb³⁺[Zn(II)MC_(pyzHA)] andillumination with the UV-A light obtained through a 377 nm band passfilter (50 nm bandwidth) for 8 min After fixation, the cells were washedand incubated with 3 μM PI for 5 min. (13A) Brightfield. (13B) NIRsignal arising from Yb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band passfilter with 60 nm bandwidth, λ_(em): 805 nm longpass filter, exposuretime: 8 s). (13C) Visible signal arising from PI (λ_(ex): 550 nm bandpass filter with 25 nm bandwidth, λ_(em): 605 nm band pass filter with70 nm bandwidth, exposure time: 800 ms). (13D) Merged image obtained bythe combination of the PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (13E)Merged image obtained by the combination of the PI,Yb³⁺[Zn(II)MC_(pyzHA)] and brightfield images. 63× objective.

FIGS. 14A to 14E illustrate results of epifluorescence microscopyexperiments on HeLa cells fixed with 150 μM Yb³⁺[Zn(II)MC_(pyzHA)] andillumination with the UV-A light obtained through a 377 nm band passfilter (50 nm bandwidth) during 10 min. After fixation, the cells werewashed and incubated with 3 μM PI for 5 min. (14A) Brightfield. (14B)NIR signal arising from Yb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band passfilter with 60 nm bandwidth, λ_(em): 805 nm longpass filter, exposuretime: 8 s). (14C) Visible signal arising from PI (λ_(ex): 550 nm bandpass filter with 25 nm bandwidth, λ_(em): 605 nm band pass filter with70 nm bandwidth, exposure time: 800 ms). (14D) Merged image obtained bythe combination of the PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (14E)Merged images obtained by the combination of the PI,Yb³⁺[Zn(II)MC_(pyzHA)] and brightfield images. 63× objective.

FIGS. 15A to 15E illustrate results of the epifluorescence microscopyexperiments on HeLa cells fixed with 15 μM concentration ofYb³⁺[Zn(II)MC_(pyzHA)] and illumination with the UV-A light during 8 minfollowed by the washing with Opti-MEM cell culture medium and by theincubation with 3 μM PI during 5 min. (15A) Brightfield. (15B) NIRsignal arising from Yb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band passfilter with 60 nm bandwidth, λ_(em): 805 nm longpass filter, exposuretime: 8 s). (15C) Visible signal arising from PI (λ_(ex): 550 nm bandpass filter with 25 nm bandwidth, λ_(em): 605 nm band pass filter with70 nm bandwidth, exposure time: 800 ms). (15D) Merged image obtained bythe combination of the PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (15E)Merged image obtained by the combination of the PI,Yb³⁺[Zn(II)MC_(pyzHA)] and brightfield images. 63× objective.

FIG. 16 illustrate results of the epifluorescence microscopy experimentson HeLa cells fixed with 30 μM concentration of Yb³⁺[Zn(II)MC_(pyzHA)]and illumination with the UV-A light during 8 min followed by thewashing with Opti-MEM cell culture medium and by the incubation with 3μM PI during 5 min. (16A) Brightfield. (16B) NIR signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band pass filter with 60 nmbandwidth, λ_(em): 805 nm long pass filter, exposure time: 8 s). (16C)Visible signal arising from PI (λ_(ex): 550 nm band pass filter with 25nm bandwidth, λ_(em): 605 nm band pass filter with 70 nm bandwidth,exposure time: 800 ms). (16D) Merged image obtained by the combinationof the PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (16E) Merged image obtainedby the combination of the PI, Yb³⁺[Zn(II)MC_(pyzHA)] and brightfieldimages. 63× objective.

FIG. 17 illustrate results of the epifluorescence microscopy experimentson HeLa cells fixed with 60 μM concentration of Yb³⁺[Zn(II)MC_(pyzHA)]and illumination with the UV-A light during 8 min followed by washingwith Opti-MEM cell culture medium and incubation with 3 μM PI during 5min. (17A) Brightfield. (17B) NIR signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] (λ_(ex): 447 nm band pass filter with 60 nmbandwidth, λ_(em): 805 nm longpass filter, exposure time: 8 s). (17C)Visible signal arising from PI (λ_(ex): 550 nm band pass filter with 25nm band width, λ_(em): 605 nm band pass filter with 70 nm bandwidth,exposure time: 800 ms). (17D) Merged image obtained by the combinationof PI and Yb³⁺[Zn(II)MC_(pyzHA)] images. (17E) Merged image obtained bythe combination of PI, Yb³⁺[Zn(II)MC_(pyzHA)] and brightfield images.63× objective.

FIGS. 18A to 18E illustrate results of NIR epifluorescence microscopy(40× magnification), of HeLa cells incubated with 150 μM ofYb³⁺[Zn(II)MC_(pyzHA)] for 12 hours, washed with OPTI-MEM® media andincubated with 3 μM propidium iodide for 5 minutes. FIG. 18A is thebrightfield image; FIG. 18B is the image of emission signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (λ_(em)=805 nm long pass filter)obtained after 8 s of exposure to excitation (λ_(ex)=447 nm band passfilter with 60 nm bandwidth); FIG. 18C is visible signal arising frompropidium iodide obtained after 80 ms of exposure time (λ_(em)=605 nmband pass filter with 70 nm bandwidth and λ_(ex)=550 nm band pass filterwith 25 nm bandwidth); FIG. 18D is FIGS. 18B and 18C merged together;and FIG. 18E is FIGS. 18A, 18B, and 18C merged together;

FIGS. 19A through 19C are confocal microscopy images (63× magnification,2× zoom) of HeLa cells incubated with 150 μM Yb³⁺[Zn(II)MC_(pyzHA)] for15 minutes, illuminated with UV light (377 nm) for 8 minutes, followedby further incubation for 1 hour (λ_(ex)=458 nm, λ_(em)=499-799 nm).FIG. 19A is the brightfield image; FIG. 19B shows the visible signalarising from Yb³⁺[Zn(II)MC_(pyzHA)]; and FIG. 19C is FIGS. 19A and 19Bmerged together;

FIGS. 20A and 20B are graphs illustrating the corrected and normalizedexcitation (λ_(em)=980 nm) and emission spectra (λ_(ex)=370 nm),respectively, of Yb³⁺[Zn(II)MC_(pyzHA)] complexes in OPTI-MEM® media andin HeLa cells, both with and without UV exposure;

FIGS. 20C and 20D are graphs illustrating the corrected and normalizedexcitation (λ_(em)=980 nm) and emission spectra (λ_(ex)=370 nm),respectively, of Yb³⁺[Zn(II)MC_(pyzHA)] complexes in cell supernatantand in HeLa cells treated with N-aceytal cysteine, both with and withoutUV exposure;

FIGS. 20E and 20F are graphs illustrating the corrected and normalizedexcitation (λ_(em)=1064 nm) and emission spectra (λ_(ex)=370 nm),respectively, of Nd³⁺[Zn(II)MC_(pyzHA)] complexes in OPTI-MEM® media andin HeLa cells, both with and without UV exposure;

FIGS. 21A to 21D illustrate optical views and associated Ramanspectroscopy mapping of CH bands intensities of (a) Live cell and cellsfixed with (b) PFA, (c) Methanol and (d) Yb³⁺[Zn(II)MC_(pyzHA)]. FIG.21E shows the associated average spectra corresponding to the part ofmaximal CH Raman signal in a-d (areas surrounded by dashed lines ina-d). The decrease of the 751 cm⁻¹ peak intensity of cytochrome c forfixed cells in respect to live cells is observed (black arrow).

FIGS. 22A through 22F are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 150 μM ofYb³⁺[Zn(II)MC_(pyzHA)] and with 3 μM propidium iodide for 15 minutes,illuminated with UV light (377 nm) for 5 minutes, further incubated for1 hour. FIG. 22A is the brightfield image; FIG. 22B is the visiblesignal arising from propidium iodide obtained after 80 ms of exposuretime (λ_(em)=605 nm 70 nm band pass filter and λ_(ex)=550 nm with 25 nmband pass filter); FIGS. 22C, 22D, and 22E show the visible signalsarising from propidium iodide and the emission signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (k_(em)=805 nm long pass filter)obtained after 8 s of exposure to excitation (λ_(ex)=447 nm band passfilter with 60 nm bandwidth) after 5, 10, 15 and 20 minutes ofincubation, respectively (in FIG. 22E, emission from PI and emissionfrom Yb³⁺[Zn(II)MC_(pyzHA)] were observed after 20 minutes); and FIG.22F is FIGS. 22A and 22E merged together;

FIGS. 23A through 23C are images (40× magnification), obtained using NIRepifluorescence microscopy, of Mesenchymal Stem Cells (MSC) incubatedwith 150 μM of Yb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, illuminated withUV light (377 nm) for 5 minutes, further incubated for 1 hour, andwashed with OPTI-MEM® media for 5 minutes. FIG. 23A is the brightfieldimage; FIG. 23B shows the emission signal arising fromYb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (λ_(em)=805 nm long pass filter)obtained after 8 s of exposure to excitation (λ_(ex)=447 nm band passfilter with 60 nm bandwidth); and FIG. 23C is FIGS. 23A and 23B merged;

FIGS. 24A through 24C are confocal microscopy images (63× magnification,2× zoom) images of MSC (Mesenchymal Stem Cells) incubated with 150 μMYb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, illuminated with UV light (377nm) for 8 minutes, followed by further incubation for 1 hour (λ_(ex)=458nm, λ_(em)=499-799 nm). FIG. 24A is the brightfield image; FIG. 24B isthe visible signal arising from Yb³⁺[Zn(II)MC_(pyzHA)]; and FIG. 24C isFIGS. 24A and 24B merged together;

FIGS. 25A through 25C are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with N-acetylcysteine (NAC) for 15 minutes, washed with OPTI-MEM® media, andincubated with 150 μM Yb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes. FIG. 25A isthe brightfield image; FIG. 25B is the emission signal image arisingfrom Yb³⁺[Zn(II)MC_(pyzHA)] in the NIR range (λ_(em)=805 nm long passfilter) obtained after 400 ms of exposure to excitation (λ_(ex)=377 nmband pass filter with 40 nm bandwidth); and FIG. 25C is FIGS. 25A and25B merged together;

FIGS. 26A through 26C are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 150 μMYb³⁺[Zn(II)MC_(pyzHA)] for 15 minutes, and then incubated with N-acetylcysteine (NAC) for 30 minutes. FIG. 26A is the brightfield image; FIG.26B is the emission signal image arising from Yb³⁺[Zn(II)MC_(pyzHA)] inthe NIR range (k_(em)=805 nm long pass filter) obtained after 5 s ofexposure to excitation (λ_(ex)=377 nm band pass filter with 40 nmbandwidth); and FIG. 26C is FIGS. 26A and 26B merged together;

FIGS. 27A through 27C are images (63× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 200 μMNd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] for 3 hours, and washed withOPTI-MEM® media. FIG. 27A is the brightfield image; FIG. 27B is theemission signal image arising from Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHAv)y)]in the NIR range (λ_(em)=805 nm long pass filter) obtained after 20 s ofexposure to excitation (λ_(ex)=536 nm band pass filter with 40 nmbandwidth); and FIG. 27C is FIGS. 27A and 27B merged;

FIGS. 28A through 28E are images (40× magnification), obtained using NIRepifluorescence microscopy, of HeLa cells incubated with 200 μMNd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] for 3 hours, washed with OPTI-MEM®media, illuminated with UV light (377 nm) for 10 minutes, furtherincubated for 1.5 hours, and incubated with 3 μM propidium iodide for 5minutes. FIG. 28A is the brightfield image; FIG. 28B shows the emissionsignal arising from Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] in the NIR range(λ_(em)=805 nm long pass filter) obtained after 20 s of exposure toexcitation (λ_(ex)=536 nm band pass filter with 40 nm bandwidth); FIG.28C shows the visible signal arising from propidium iodide obtainedafter 80 ms of exposure time (λ_(em)=617 nm band pass filter andλ_(ex)=535 nm band pass filter with 40 nm bandwidth); FIG. 28D is FIGS.28B and 28C merged; and FIG. 28E is FIGS. 28A, 28B and 28C merged;

FIGS. 29A and 29B illustrate the images of HeLa cells fixed withYb³⁺[Zn(II)MC_(pyzHA)] obtained under epifluorescence microscopy.Brightfield images of fixed cells were recorded after differentincubation time at 37° C. in Opti-MEM: (29A) 1 h and (29B) 1 month.

TABLE 1 illustrates the photophysical properties ofYb³⁺[Zn(II)MC_(pyzHA)] and Nd³⁺[Zn(II)MC_(pyzHA)] MCs in solid state andin aqueous solutions (200 μM) at room temperature.^(a) τ(μs)^(b)Q(%)^(d) Metallacrown Solid H₂O D₂O q^(c) Solid H₂O D₂OYb³⁺[Zn(II)MC_(pyzHA)] 45.6(3)  5.57(1)  81.3(1)  0   0.659(4) 1.12(7) ·10⁻² 0.257(3) Nd³⁺[Zn(II)MC_(pyzHA)]  1.71(1) 0.214(4)  1.29(1) 0.10.444(9)  7.7(1) · 10⁻³ 6.17(9) · 10⁻² ^(a)2σ values are given betweenparentheses. Experimental errors: τ, ±2%, Q, ±10%. ^(b)Under excitationat 355 nm. ^(c)The number of coordinated water molecules (q) has beencalculated using the equations:${q_{Yb} = {{\frac{1}{\tau_{H2O}} - \frac{1}{\tau_{D2O}} - {0.2\mspace{14mu} \left( {{in}\mspace{14mu} {\mu s}} \right)\mspace{14mu} {and}\mspace{14mu} q_{Nd}}} = {{130 \times \left( {\frac{1}{\tau_{H2O}} - \frac{1}{\tau_{D2O}}} \right)} - 0.4}}};$[274, 275] estimated error ±0.2. ^(d)Under excitiation at 370 nm.

DESCRIPTION

The present invention relates to a method for simultaneously fixing andstaining cells, preferably in vitro, the method comprising:

-   initially incubating the cells in a solution including a    Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex, wherein the HA ligand    is a hydroximate ligand;-   exposing the incubating cells to ultraviolet (UV) light; and-   continuing to incubate the cells in the solution after UV light    exposure.

The present invention involves the use of a Ln(III)Zn₁₆(HA ligand)₁₆metallacrown complex, wherein the HA ligand is a hydroximate ligand.

The metallacrown complexes used according to the invention may also berepresented by the general formula Ln³⁺[Zn(II)MC_(HA)], wherein the HAligand is a hydroximate ligand.

The preferred HA ligand will be described in detail hereafter.Preferably, the HA ligand is pyrazinehydroximate.

According to an embodiment, the metallacrown complex according to theinvention may be represented by the following formula:Ln³⁺[Zn(II)MC_(pyzHA)].

According to an embodiment, the above-mentioned solution includes theLn(III)Zn₁₆(HA ligand)₁₆ metallacrown complex in a medium; and aconcentration of the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex inthe solution ranges from about 90 μM to about 400 μM.

According to an embodiment, the method according to the inventionfurther comprises culturing the cells prior to incubating the cells inthe solution.

Preferably, the method according to the invention comprises thefollowing steps:

initially incubating the cells is accomplished for a time (T1) rangingfrom about 10 minutes to about 3 hours;

exposing the incubating cells to UV light is accomplished for a time(T2) ranging from about 5 minutes to about 10 minutes; and

continuing to incubate the cells is accomplished for a time (T3) rangingfrom about 1 hour to about 2 hours.

Preferably, the method according to the invention further comprisesexposing the incubating cells to additional UV light for a time (T4)ranging from about 1 minute to about 5 minutes.

According to a preferred embodiment, the HA ligand of the Ln(III)Zn₁₆(HAligand)₁₆ metallacrown complex is selected from the group consisting ofpyrazinehydroximate, quinoxalinehydroximate, quinaldinehydroximate, andcombinations thereof.

Preferably, the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex is one of:

[Ln(III)Zn₁₆(pyrazinehydroximate)₁₆(pyridine)₈]counter ion;

[Ln(III)Zm₆(quinoxalinehydroximate)₁₆(pyridine)₈]counter ion; or

[Ln(III)Zn₁₆(quinaldinehydroximate)₁₆(pyridine)₈] counter ion;

the Ln(III) is selected from the group consisting of Y³⁺, La³⁺, Ce³⁺,Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺,and Lu³⁺; and

the counter ion is selected from the group consisting of a triflate, amesylate, a besylate, a camsylate, an edisylate, an estolate, anesylate, a napsylate, a tosylate, a fluoride, a chloride, a bromide, aniodide, a nitrate, a sulfate, a carbonate, an acetate, a phosphate, anda sulfonate.

According to an embodiment, the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrowncomplex includes a mixture of metallacrown complexes; each species inthe metallacrown mixture is[Ln(III)Zn₁₆(pyrazinehydroximate)_(x)(quinoxalinehydroximate)_(y)(pyridine)₈]counter ion, wherein x+y=16, wherein preferably x ranges from 8 to 13and y ranges from 3 to 8;

the Ln(III) is selected from the group consisting of Y³⁺, La³⁺, Ce³⁺,Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺,and Lu³⁺; and

the counter ion is selected from the group consisting of a triflate, amesylate, a besylate, a camsylate, an edisylate, an estolate, anesylate, a napsylate, a tosylate, a fluoride, a chloride, a bromide, aniodide, a nitrate, a sulfate, a carbonate, an acetate, a phosphate, anda sulfonate.

According to an embodiment, the above-mentioned medium is aserum-supplemented medium.

According to a preferred embodiment, in the method according to theinvention, UV light exposure induces death of at least some of thecells; and after continuing to incubate the cells, the Ln(III)Zn₁₆(HAligand)₁₆metallacrown complex is located in nuclei and cytoplasm ofleast some dead cells.

The present invention also relates to an optical imaging method,preferably in vitro, comprising:

forming simultaneously fixed and stained cells by:

-   -   initially incubating cells in a solution including a        Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex, wherein the HA        ligand is a hydroximate ligand;    -   exposing the incubating cells to ultraviolet (UV) light; and    -   continuing to incubate the cells in the solution after UV light        exposure; and

exposing the simultaneously fixed and stained cells to an opticalimaging technique selected from the group consisting of epifluorescencemicroscopy, confocal microscopy, and combinations thereof.

According to an embodiment, the optical imaging method as mentionedabove further comprising any of:

tuning an excitation response of the simultaneously fixed and stainedcells by changing the Ln(III) of the Ln(III)Zn₁₆(HAligand)₁₆metallacrown complex; or

tuning an emission response of the simultaneously fixed and stainedcells by changing the hydroximate ligand of the Ln(III)Zn₁₆(HA ligand)₁₆metallacrown complex.

The present invention also relates to a method for selective labellingof necrotic cells, preferably in vitro, the method comprising incubatingthe cells in a solution including a Ln(III)Zn₁₆(HA ligand)₁₆metallacrown complex, wherein the HA ligand is a hydroximate ligand.

According to an embodiment, the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrowncomplex is as defined above.

DETAILED DESCRIPTION

In the example methods disclosed herein, lanthanide-based metallacrowncomplexes are used as probes for necrotic cells. In particular, thelanthanide-based metallacrown complexes operate as NIR and/or visiblestains for the nucleus and the cytoplasm of the necrotic cells. Inaddition, the present inventors have found that a combination of shortexposure to ultraviolet (UV) light and a high concentration of thelanthanide-based metallacrown complex brings about a photochemicaleffect, which is similar to cell fixation that may be obtained usingformaldehyde or methanol. Fixing the dead cells in the manner disclosedherein preserves the morphology of the dead cells, and thus the fixedcells do not dissolve or decay.

As such, in the examples disclosed herein, the cells are simultaneouslystained and fixed. “Simultaneously stained and fixed” means that thecells are exposed to counter staining and undergo fixation during thesame procedure, and the acts of staining and fixing may occur at exactlythe same time. Examples of the staining and mixing method will bediscussed in more detail below.

As mentioned above, lanthanide-based metallacrown complexes are utilizedas optical imaging agents/probes and cell fixation agents.Lanthanide(III) metal ions (Ln(III) or Ln³⁺) contain 4f orbitals andexhibit unique luminescent characteristics. However, most f-ftransitions of Ln³⁺ ions are forbidden by quantum mechanics rulesinducing low absorption coefficients, resulting in inefficient directexcitation. As such, use of the lanthanide signal in optical imaging isuncommon. In the examples disclosed herein, the structure of thelanthanide-based metallacrown complex may be controlled during itsformation to provide optimized sensitization of the lanthanide cation,as well as protection against non-radiative deactivations.

The lanthanide-based metallacrown complexes disclosed herein arethree-component supramolecular assemblies with transition metals,tetra-dentate ligands that are capable of transferring energy to acentral ion and/or producing a ligand-based charge transfer state whenincorporated into the metallacrown complex, and lanthanide ions (Ln³⁺).The tetra-dentate ligands cyclize to form a repeating [-M-N—O—]_(x)sub-unit, where M is the transition metal ion. Similar to crown ethers,metallacrowns can be synthesized with a range of sizes, and the inwardfacing oxime oxygen atoms are capable of binding to a central metal ion.

As mentioned above, the structure of the lanthanide-based metallacrowncomplex may be controlled to achieve a desired response. For example, bychanging the ligand of the lanthanide-based metallacrown complex, theexcitation response of the metallacrown complex may be altered. Foranother example, by changing the lanthanide metal, the emission responseof the metallacrown complex may be altered. Characteristic NIR emission(i.e., NIR luminescence properties) of the lanthanide metal ions in thelanthanide-based metallacrown complexes disclosed herein may be observedunder excitation wavelengths ranging from the UV to the visible range.

In the examples disclosed herein, the lanthanide-based metallacrowncomplexes may be referred to as Ln(III)TM₁₆(HA ligand)_(m)metallacrowncomplexes, where Ln(III) is the lanthanide metal ion, TM is thetransition metal ion, and HA ligand is the hydroximate ligand. Thechemical formula of the metallacrown complex includes at least 1lanthanide metal ion, 16 transition metal ions and 16 HA ligands. The 16transition metal ions and 16 HA ligands are distributed among twocapping crowns (i.e., [12-MC-4]₂) and an encapsulating crown (i.e.,[24-MC-8]) which together form the entire metallacrown complex. Usingmetallacrown nomenclature, the lanthanide-based metallacrown complexesmay be shown as Ln(III)[12-MC-4]₂[24-MC-8]. “MC” refers to themetallacrown macrocycle with a repeating sub-unit consisting of thetransition metal ion (M(II)) and the hydroximate (HA) ligand.

The Ln(III) is a central ion that bonds (e.g., via coordination bonding)to the capping crowns, [12-MC-4]. It is to be understood that Ln³⁺ mayinclude any lanthanide ion, such as dysprosium (Dy³⁺), ytterbium (Yb³⁺),neodymium (Nd³⁺), gadolinium (Gd³⁺), terbium (Tb³⁺), europium (Eu³⁺),erbium (Er³⁺), lanthanum (La³⁺), cerium (Ce³⁺), praseodymium (Pr³⁺),promethium (Pm³⁺), samarium (Sm³⁺), holmium (Ho³⁺), thulium (Tm³⁺), orlutetium (Lu³⁺). Throughout the application, the “Ln³⁺”, “Ln(III)”, or“lanthanide” may be replaced with other rare-earth metal ions, such asyttrium (Y³⁺) and scandium (Sc³⁺). FIG. 1 illustrates the emissionspectra of luminescent lanthanides. This figure clearly illustrates thesharp emission bands of each lanthanide, and that there is spectraldiscrimination among the various lanthanides.

The transition metal ion (TM(II)) may be selected from the groupconsisting of Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Rh²⁺, Pd²⁺, Ag²⁺, Cd²⁺, Ir²⁺,Pt²⁺, Au²⁺, or Hg²⁺.

The HA ligand may be pyrazinehydroximate, quinoxalinehydroximate,quinaldinehydroximate, or combinations thereof. Pyrazinehydroximate(pyzHA) has the structure:

and is formed from the deprotonation of pyrazinehydroxamic acid:

Similarly, quinoxalinehydroximate (quinoHA, not shown) is formed fromthe deprotonation of quinoxalinehydroxamic acid:

and quinaldinehydroximate (quinHA, not shown) is formed from thedeprotonation of quinaldinehydroxamic acid (sometime referred to asquinaldichydroxamic acid):

Derivatives of the previously listed hydroxamic acids (structures 2, 3,4) may also be used to form suitable hydroximate ligands. For example,any combination of R-groups may be bound to positions 3, 5 and 6 inStructure 2, to positions 3 and 5-8 in Structure 3, and to positions 3-8in Structure 4 above (where position 1 is the N atom at the top of thering the numbered positions proceed to the right around the ring(s), andwherein the carbon atoms in Structures 3 and 4 that are shared betweenthe two rings are not numbered). In an example, the R-groups areindependently selected from —H, -D (deuterium), —OH, —SH, —NH₂, —NO₂,—F, —Cl, —Br, —I, —CF₃, —OCH₃, —SO₃H, —CH₃, and —CN. In another example,the R-group may also be a fused aromatic ring. Examples of the fusedaromatic ring include benzene, naphthalene, phenanthrene, chrysene, orpyrene. It is to be understood that each position on the fused aromaticring may also have R-groups bound thereto (e.g., —H, -D (deuterium),—OH, etc.). In yet a further example, the R-group at any of positions3-8 of Structure 1 may also be a fused heterocyclic ring. Examples ofthe fused heterocyclic ring include furan, thiophene, pyrrole, pyridine,imidazole, thiazole, pyrimidine, indole, isoindole, indolizine, purine,carbazole, dibenzofuran, oxazole, or isoxazole. It is to be understoodthat each position on the fused heterocycle may also have R-groups boundthereto (e.g., —H, -D (deuterium), —OH, etc.). In still another example,the R-group(s) may be amides, or chromophoric or recognition regions,such as biotin, sugar, oligos, peptides (e.g., RGD), antibodies, or thelike. In still other examples, the R groups may be ═O, ═N, —N₃, —NR′H,—NR′₂, —NR′³⁺, —COOH, —COOR′, —CH₂—R′, —CHR₂, —CHR′R″, —CR′R″R′″, —OR′,or the like, where R′, R″, and R′″ may be independently selected fromany of the aforementioned R-groups.

Examples of other derivatives include heterocycles derived from thehydroxamic acids with carbon (instead of nitrogen) at position 4 inStructures 2 and 3, or nitrogen, oxygen or sulfur (instead of carbon) atpositions 3 to 8 in Structure 4. It is to be understood that thesederivatives could contain nitrogen, oxygen or sulfur atoms individuallyor in combination at the various positions.

The HA ligands disclosed herein provide the complexes with a uniquemolecular structure. In addition, this ligand or a derivative thereofefficiently absorbs excitation light and transfers the resulting energyto the Ln(III) ion. It has been unexpectedly found that the metallacrowncomplexes formed with the HA ligand(s) or a derivative thereof provide acombination of a near-visible ligand-based charge transfer absorption,remarkably high quantum yields (exhibiting intense near-infraredluminescence), and long luminescence lifetimes. Complexes with theseproperties may be used in a variety of applications, including theexample simultaneous cell staining and imaging methods disclosed herein.

As mentioned above, the metallacrown complexes include the two cappingcrowns, [12-MC-4]. Example structures of the [12-MC-4] capping crownsare shown below, with 50% thermal displacement parameters and a partialnumbering scheme:

Structures 5 and 6 illustrate the Ln(III) ion (in these examples Dy)bonded to the four inward-facing hydroximate oxygen atoms of eachcapping crown. In Structure 5, these inward-facing hydroximate oxygenatoms are labeled O6, O6A, O6B and O6C, and in Structure 6, theseinward-facing hydroximate oxygen atoms are labeled O4, O4A, O4B and O4C.During the method of making the metallacrown complex (discussed below),the HA ligand cyclizes to form a repeating [M(II)HA] sub-unit, which in[12-MC-4], is repeated four times. As a result, each [12-MC-4] cappingcrown has twelve total atoms (e.g., 4 Zn, 4 O, and 4 N) in itsmacrocyclic ring.

The metallacrown complex also includes the encapsulating crown,[24-MC-8]. An example structure of the [24-MC-8] encapsulating crown isshown below, with 50% thermal displacement parameters and a partialnumbering scheme:

In this example, the HA ligand cyclizes to form the repeating [M(II)HA]sub-unit, which in [24-MC-8] is repeated eight times. As a result, each[24-MC-8] encapsulating crown has twenty-four total atoms (i.e., 8 Zn, 8O, and 8 N) in its macrocyclic ring.

Structure 7 also illustrates the non-bonded central Ln(III) ion (e.g.,Dy1). The dashed lines indicate how the central crown bridges to thesmaller, concave, capping crowns (Structures 5 and 6).

The method used to form the metallacrown complex may utilize pyridine orsome other solvent (e.g., dimethylformamide or methanol), and thus theencapsulating crown (Structure 7) may also have eight pyridine rings, orsome other coordinating solvent molecules, attached thereto.

As mentioned above, the transition metal may be any divalent transitionmetal (II) ion. In the examples disclosed herein, Zn²⁺ is the transitionmetal ion used. The coordination geometry of Zn²⁺ in [24-MC-8] (A) andin [12-MC-4] (B) is shown below:

The coordination geometry of Zn²⁺ indicates that the distinctmetallacrown rings are linked through bridging oxygen atoms, namely the[12-MC-4] carboxylate oxygen atoms (identified with solid arrows) andthe [24-MC-8] hydroximate oxygen atoms (identified with dashed arrows).In other words, the [24-MC-8] unit has a cavity at its center, and thesandwich complex formed between the Ln³⁺ ion and the two [12-MC-4] unitsis bound within that cavity through the bridging oxygen atoms. Asillustrated in (B) above, the Ln³⁺ ion is bound to the [12-MC-4]hydroximate oxygen atoms (identified by dotted arrows).

A charge balance of the metallacrown complex that is formed may beobtained by the presence of a negatively charged species, such as anunbound counterion (e.g., triflate(s), mesylate(s), besylate(s),camsylate(s), edisylate(s), estolate(s), esylate(s), napsylate(s),tosylate(s), fluoride(s), chloride(s), bromide(s), iodide(s),nitrate(s), sulfate(s), carbonate(s), acetate(s), sulfonate(s), orphosphate(s)).

Specific examples of the Ln(III)TM₁₆(HA ligand)₁₆metallacrown complexesinclude: [Ln(III)Zn₁₆(pyrazinehydroximate)₁₆(pyridine)₈]counter ion;[Ln(III)Zn₁₆(quinoxalinehydroximate)₁₆(pyridine)₈]counter ion; or[Ln(III)Zn₁₆(quinaldinehydroximate)₁₆(pyridine)₈]counter ion. As anotherexample, the Ln(III)TM₁₆(HA ligand)₁₆ metallacrown complex may be amixed ligand metallacrown complex, with at least two different HAligands. When mixed ligand metallacrown complexes are formed, the resultmay be a mixture of metallacrown complexes, where each species in themetallacrown mixture is [Ln(III)TM₁₆(HA ligand 1)_(x)(HA ligand2)_(y)(pyridine)₈]counter ion, wherein x+y=16, and wherein preferably xranges from 8 to 13, and thus y ranges from 8 to 3. As a more specificexample, the mixture may include

-   [Ln(III)Zn₁₆(pyrazinehydroximate)_(x)(quinoxalinehydroximate)_(y)(pyridine)₈]counter    ion, where the predominant species is x=12 and y=4 (e.g.,-   [Nd(III)Zn₁₆(pyrazinehydroximate)₁₂(quinoxalinehydroximate)₄(pyridine)₈](CF₃SO₃)),    but other species (in which x=13 and y=3, and x=11 and y=5) may also    be present.

In an example of the method for making the lanthanide-based metallacrowncomplex, an example of a hydroxamic acid precursor of the HA ligand, atransition metal salt, and a rare-earth metal salt are dissolved in asolvent to form a solution. In another example of the method for makingthe lanthanide-based metallacrown complex, the hydroxamic acid precursorof the HA ligand is dissolved in the solvent to form a solution, andthen the transition metal salt and the rare-earth metal salt are added.Example solvents include dimethylformamide (DMF), methanol, pyridine,water, and combinations thereof.

The hydroxamic acid precursor of the HA ligand may be prepared in asingle step. In an example, fresh hydroxylamine is first prepared bycombining hydroxylamine hydrochloride and potassium hydroxide inmethanol at about 0° C. This solution may be stirred (e.g., for about 20minutes or longer) and filtered to remove potassium chloride. Quinaldicacid and N-methylmorpholine are combined with stirring indichloromethane. This solution may be cooled to about 0° C., at whichtime ethylchloroformate is added. This reaction may be stirred for about20 minutes to 1 hour, and then filtered. The hydroxylamine solution isadded to the filtrate at about 0° C. This reaction mixture may beallowed to warm to room temperature and stirred for about 1.5 hours. Thevolume may then be reduced to about 200 mL en vacuo and water is addedto induce the precipitation of a white solid. The solid is collected byfiltration, and may be triturated with hot (about 40° C.)dichloromethane to yield quinaldichydroxamic acid as a white powder. Thehydroxamic acid may be used in the synthesis of the MC complexes. Duringthe synthesis, the hydroxamic acid will undergo deprotonation.

Any transition metal salt may be used. In an example, the transitionmetal salt is a triflate (—Otf), a mesylate, a besylate, a camsylate, anedisylate, an estolate, an esylate, a napsylate, a tosylate, a fluoride,a chloride, a bromide, an iodide, a nitrate, a sulfate, a carbonate,e+2+, _(Ni2+, Cu)2+_(, zn)2+_(, Rh)2+_(, pd)2+_(, Ag) 2+_(, Cd)2+_(,) anacetate, a sulfonate, or a phosphate of any of Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺,Rh²⁺, Pd²⁺, Ag²⁺, Cd²⁺, Ir²⁺, Pt²⁺, Au²⁺, or Hg²⁺.

The rare-earth metal salt may be a triflate, a mesylate, a besylate, acamsylate, an edisylate, an estolate, an esylate, a napsylate, atosylate, a fluoride, a chloride, a bromide, an iodide, a nitrate, asulfate, a carbonate, an acetate, a sulfonate, or a phosphate of any ofY³⁺, Sc³⁺, La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺,Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺, or Lu³⁺.

As mentioned above, in some examples of the method, the precursor to theHA ligand, the transition metal salt, and the rare-earth metal salt aredissolved in the solvent to form a solution. This example method may besuitable when quinaldinehydroxamic acid is used as the precursor to theHA ligand. In this example method, a base is then added to the solution.Examples of suitable bases include triethylamine (TEA), trimethylamine,or other Bronsted bases. In an example, when the base is added to thesolution, the resulting reaction mixture turns yellow.

In this example method, the reaction mixture (i.e., the solution and thebase) is then stirred for a predetermined time at a predeterminedtemperature. In an example, the temperature is room temperature (e.g.,from about 18° C. to about 22° C.) and the time ranges from about 12hours to about 24 hours.

The reaction mixture is then exposed to a purification method to producethe highly pure metallacrown complex. Examples of suitable purificationmethods include recrystallization by slow evaporation of the solvent,recrystallization by vapor diffusion, recrystallization by solventlayering, high-performance liquid chromatography (HPLC), or flashchromatography. In one example, pyridine is used in the purificationprocess, which results in the introduction of pyridine ligands to themetallacrown complex.

Also as mentioned above, in some other examples of the method, theprecursor to the HA ligand is dissolved in the solvent to form asolution. This example method may be suitable when pyrazinehydroxamicacid and/or quinoxalinehydroxamic acid are used as the precursor(s) tothe HA ligand(s).

In this example method, the HA ligand is dissolved in the solvent toform a solution. The solution may be stirred in order to completelydissolve the solid. The transition metal salt and the rare-earth metalsalt may then be added to the solution. The solution may be stirred atroom or an elevated temperature) for a predetermined amount of time, andthen filtered.

The following are three examples of this example of the method:

Synthesis of [Zn₁₆Ln(pyzHA)₁₆(py)₈](OTf)₃

Pyrazinehydroxamic acid (45.0 mg, 0.32 mmol) is added into a solutioncontaining 1.0 mL pyridine and 10.0 mL water. The solution is stirredfor about 5 minutes to completely dissolve the solid. Zinc triflate(116.0 mg, 0.32 mmol) and Lanthanide triflate (0.04 mmol) are added, andthe yellow solution is stirred for about 5 minutes at room temperatureand then filtered. The filtrate may be left undisturbed in order toyield yellow crystals after three days. The crystals may be collected byfiltration and dried in air.

Synthesis of [Zn₁₆Ln(quinoHA)₁₆(py)₈](OTf)₃

Quinoxalinehydroxamic acid (60.5 mg, 0.32 mmol) is added into a solutioncontaining 1.0 mL pyridine, 5.0 mL DMF, and 5.0 mL water. The solutionis stirred for about 5 minutes to completely dissolve the solid. Zinctriflate (116 mg, 0.32 mmol) and Lanthanide triflate (0.04 mmol) areadded, and the orange solution is stirred at 80° C. for about 2 hours,cooled down to room temperature, and then filtered. The filtrate may beleft undisturbed to yield red crystals after one week. The crystals maybe collected by filtration, washed with water, and dried in air.

Synthesis of [Zn₁₆Ln(pyzHA)_(x)(quinoHA)_(y)(py)₈](OTf)₃

Pyrazinehydroxamic acid (37.5 mg, 0.27 mmol) and Quinoxalinehydroxamicacid (17.0 mg, 0.09 mmol) are added into a solution containing 1.0 mLpyridine and 5.0 mL water. The solution is stirred for 5 minutes tocompletely dissolve the solids. Zinc triflate (131.0 mg, 0.36 mmol) andLanthanide triflate (0.045 mmol) are added and the orange solution isstirred at room temperature for about 20 minutes, and then filtered. Thefiltrate may be left undisturbed to yield a mixture of yellow/redcrystals after one week. The crystals are collected by filtration,washed with water, and dried in air.

The metallacrown complex may be used in examples of the methodsdisclosed herein. One example method is for simultaneously counterstaining and fixing of cells and another example method is an opticalimaging method for necrotic cells.

In an example of the method for simultaneously staining and fixingcells, the cells may first be obtained and prepared. Examples ofsuitable cells include human epithelial cervix carcinoma (HeLa) cells,mesenchymal stem cells (MSC), or other suitable cells. Preparation ofthe cells may involve seeding and culturing the cells. It is to beunderstood that the cell culture medium selected may vary, dependingupon the cell line that is utilized. The cells may be washed with freshmedium.

Throughout the steps of the method, at least some of the cells (e.g., atleast 90%), and in many instances, all of the cells, undergo necrosis(unprogrammed cell death) These cells may also be a combination ofnecrotic cells and apoptotic cells.

The cells are incubated (initially) in a solution including theLn(III)TM₁₆(HA ligand)₁₆ metallacrown complex(es) in a medium.Throughout the discussion of this example method, the Ln(III)TM₁₆(HAligand)₁₆ metallacrown complex will be referred to as theLn³⁺[Zn(II)MC_(HA)] metallacrown complex(es). It is to be understoodthat the medium selected may vary, depending upon the cell line that isutilized. The medium may be a serum-supplemented reduced medium (such asOPTI-MEM® media supplemented with fetal bovine serum (FBS)). Theconcentration of the Ln³⁺[Zn(II)MC_(HA)] metallacrown complex in thesolution may range from about 45 μM to about 400 μM. As specificexamples, the concentration of the Ln³⁺[Zn(II)MC_(HA)] metallacrowncomplex of the solution may be about 45 μM, 150 μM or 200 μM.

The initial incubation may take place for a predetermined time,depending on the cell line and metallacrown complex that are used. Theinitial incubation time (T1) may range from about 10 minutes to about 3hours. As examples, a metallacrown complex formed with Yb³⁺ and withNd³⁺ may be initially incubated for about 15 minutes.

During the initial incubating period, at least some of the cells maydie. The percentage of cells that die during this point of the methodmay depend upon the concentration of the Ln³⁺[Zn(II)MC_(HA)]metallacrown complex and the initial incubation time (T1). When theinitial incubation time (T1) is less than 24 hours, and theconcentration ranges from about 2.5 μM to about 100 μM, up to 10% of thecells may die.

The incubating cells are then exposed to UV light. In an example, the UVlight is UV-A light. UV light exposure may take place for a time (T2)ranging from about 5 minutes to about 10 minutes. The short exposure toUV light, in combination with the high concentration of theLn³⁺[Zn(II)MC_(pyzHA)] metallacrown complex brings about a photochemicaleffect, which is similar to cell fixation that may be obtained usingformaldehyde or methanol.

After UV light exposure, the cells are allowed to continue to incubatein the solution. In an example, the continued incubation of the cells inthe solution occurs for a time (T3) ranging from about 1 hour to about 2hours. Continued cell incubation after UV light exposure allows theLn³⁺[Zn(II)MC_(HA)] metallacrown complex to stain the nucleus and thecytoplasm of at least some (if not all) of the fixed cells.

In some examples of the method, the incubating cells are not exposed toany additional UV light. In some other examples of the method, theincubating cells may be exposed to additional UV light for a time (T4)ranging from about 1 minute to about 5 minutes.

After continued incubation, and if performed, additional UV lightexposure, the stained and fixed cells may be washed (e.g., with freshmedium). The simultaneously stained and fixed cells may be an opticalimaging technique, such as epifluorescence microscopy, confocalmicroscopy, or combinations therefore.

An example of the optical imaging method disclosed herein includesforming the simultaneously fixed and stained cells via the methoddisclosed herein, and exposing the simultaneously fixed and stainedcells to the optical imaging technique.

To further illustrate the present disclosure, examples are given herein.It is to be understood that these examples are provided for illustrativepurposes and are to be construed as non-limiting.

EXAMPLES

In the following examples, Yb³⁺[Zn(II)MC_(pyzHA)] complexes (which canalso be described as YbZn₁₆(pyz)₁₆MC), Nd³⁺[Zn(II)MC_(pyzHA)] (which canalso be described as NdZn₁₆(pyz)₁₆MC) complexes, orNd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] (NdZn₁₆(pyz)_(x)(quino)_(y)MC)complexes are utilized. The respective complexes were formed by thefollowing procedures:

Synthesis of Yb³⁺[Zn(II)MC_(pyzHA)] complex or Nd³⁺[Zn(II)MC_(pyzHA)]Complex

Pyrazinehydroxamic acid (45.0 mg, 0.32 mmol) was added into a solutioncontaining 1.0 mL pyridine and 10.0 mL H₂O. The solution was stirred for5 minutes to completely dissolve the solid. Zinc triflate (116.0 mg,0.32 mmol) and Ytterbium triflate or Neodymium triflate (0.04 mmol) wereadded, and the yellow solution was stirred for 5 minutes at roomtemperature and then filtered. The filtrate was left undisturbed to giveyellow crystals after three days. The crystals were collected byfiltration and dried in air.

Synthesis of Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] Complex

Pyrazinehydroxamic acid (37.5 mg, 0.27 mmol) and Quinoxalinehydroxamicacid (17.0 mg, 0.09 mmol) were added into a solution containing 1.0 mLpyridine and 5.0 mL H₂O. The solution was stirred for 5 minutes tocompletely dissolve the solids. Zinc triflate (131.0 mg, 0.36 mmol) andNeodymium triflate (0.045 mmol) were added, and the orange solution wasstirred at room temperature for 20 minutes and then filtered. Thefiltrate was left undisturbed to give a mixture of yellow/red crystalsafter one week. The crystals were collected by filtration, washed withH₂O, and dried in air.

In several of the following examples, Yb³⁺[Zn(II)MC_(pyzHA)]complexeswere formed. The left side of FIG. 2 illustrates pyrazinehydroxamicacid. The right side of FIG. 2 is a representation of the metallacrownstructure (as obtained from X-Ray structure) which is formed by thesupramolecular assembly of the hydroxamic acid units (which form the pyzHA ligands) with zinc and ytterbium salts in a controlled ratio. TheYb³⁺[Zn(II)MC_(pyzHA)]complex has a unique and rigid structure where thelanthanide ion is positioned in the center of the structure (turquoisesphere) and surrounded with zinc (grey spheres).

The structure of Yb³⁺[Zn(II)MC_(pyzHA)] complex and other MC complexesprovide sensitization and protection to the luminescent lanthanides.These metallacrowns exhibit promising photophysical properties which areamong the highest values for quantum yields and luminescence lifetimes.As will be illustrated in the following examples, several of the MCcomplexes have demonstrated remarkable NIR luminescence properties (someof which show the highest luminescence quantum yield values reported todate for selected NIR emitting lanthanides (Yb³⁺, Nd³⁺)). As shown inFIG. 4 (Top), metallacrowns formed with ytterbium can be excited up to480 nm. As will be illustrated in several of the following examples,these metallacrowns demonstrated intense near-infrared signals in cancercells for good signal to noise ratio and high sensitivity of detection.

Example 1 NIR Epifluorescent Microscopy of theYb³⁺[Zn(II)MC_(pyzHA)]Complex with HeLa Cancer Cells or Mesenchymal StemCells (MSC)

Experimental Conditions

HeLa (Human Epithelial Cervix Carcinoma) cell line, obtained from ATCC(Molsheim, France), was cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS),1% of 100× non-essential aminoacid solution, 1% of L-glutamine(GlutaMAX) and 1% of streptomycin/penicillin antibiotics. Cells wereseeded in a 8-well Lab Tek Chamber coverglass (Nunc, Dutsher S.A.,Brumath, France) at a density of 6×10⁴ cells/well and cultured at 37° C.in 5% humidified CO₂ atmosphere.

The MSC (Mesenchyml Stem Cells) cell line was obtained from Universityin Orleans, 13MTO Laboratory (Orleans, France). The as-received MSC cellline has been obtained from the bone marrow of rats. The MSC cell linewas cultured in Minimum Essential Medium (MEM) supplemented with 10%heat-inactivated fetal bovine serum (FBS), 1% of L-glutamine (GlutaMAX)and 1% of streptomycin/penicillin antibiotics. The MSC were also seededin a similar manner to the HeLa cells.

After 24 hours, the respective cell culture media was removed and theHeLa cells or MSC were incubated with the Yb³⁺[Zn(II)MC_(pyzHA)]complexaccording to one of the following procedures.

Long incubation, no UV light exposure: Some of HeLa cells were washedtwice with OPTI-MEM® media (room temperature), and were incubated with asolution of 150 μM Yb³⁺[Zn(II)MC_(pyzHA)]complex in OPTI-MEM® media(supplemented with 2% of FBS at 37° C. in 5% CO₂ atmosphere) for 12hours.

Short incubation, UV light exposure #1: Some of the HeLa cells werewashed twice with OPTI-MEM® media (room temperature), incubated with asolution of 150 μM Yb³⁺[Zn(II)MC_(pyzHA)] complex in OPTI-MEM® media(supplemented with 2% of FBS at 37° C. in 5% CO₂ atmosphere) for 15minutes, illuminated with UV light (377 nm) for 8 minutes, and then wereallowed to continue incubating for 1 hour. The continued incubationenabled the internalization of the complexes.

Short incubation, no UV light exposure: Some of HeLa cells were washedtwice with OPTI-MEM® media (room temperature), and were incubated with asolution of 45 uM Yb³⁺[Zn(II)MC_(pyzHA)]complex in OPTI-MEM® media(supplemented with 2% of FBS at 37° C. in 5% CO₂ atmosphere) for 15minutes.

All of the previously mentioned incubated HeLa cells were then washedwith fresh OPTI-MEM® media and were incubated with 3 uM propidium iodidefor 5 minutes. Propidium iodide is a commercially available stain(emitting in the visible range) for necrotic cells, and was used toconfirm cell necrosis.

Short incubation, UV light exposure #2: Some of the HeLa cells werewashed twice with OPTI-MEM® media (room temperature), incubated with asolution of 150 μM Yb³⁺[Zn(II)MC_(pyzHA)]complex in OPTI-MEM® media(supplemented with 2% of FBS at 37° C. in 5% CO₂ atmosphere) and 3 μMpropidium iodide for 5 minutes, illuminated with UV light (377 nm) for 5minutes, and then were allowed to continue incubating for 1 hour. Thecontinued incubation enabled the internalization of the complexes.

Short incubation, UV light exposure #3: The MSC cells were incubatedwith a solution of 150 μM Yb³⁺[Zn(II)MC_(pyzHA)]complex in OPTI-MEM®media (supplemented with 2% of FBS at 37° C. in 5% CO₂ atmosphere) for15 minutes, illuminated with UV light (377 nm) for 8 minutes, and thenwere allowed to continue incubating for 1 hour.

Prior to epifluorescent imaging, the HeLa and the MSC cells were washedtwice with OPTI-MEM® (room temperature) in order to remove anynon-specifically bound Yb³⁺[Zn(II)MC_(pyzHA)] complex. The cells wereobserved with a Zeiss Axio Observer Z1 fluorescence inverted microscope(Zeiss, Le Pecq, France) equipped with an EMCCD Evolve 512 (RoperScientific) photometric camera or ORCA-R2 high resolution CCD camera.The light source, Zeiss HXP 120, was combined with the following filtercubes:

-   i. 377 nm with 50 nm band-pass filter with 50 nm bandwidth for the    excitation and 805 nm long-pass filter 805 nm or 996 nm band-pass    filter with 70 nm bandwidth for YbIII emission in the NIR range.-   ii. 44780 nm band-pass filter with 650 nm bandwidth for the    excitation and 805 nm long-pass filter 805 nm or 996 nm band-pass    filter with 70 nm bandwidth for YbIII emission in the NIR range.

Experimental Results

The results shown in FIGS. 18A-18E are for the cells with the longincubation and no UV light exposure. The results in FIG. 18B indicatethat the incubation of HeLa cancer cells withYb³⁺[Zn(II)MC_(pyzHA)]leads to cell death. From these results, it can beconcluded that the MC is toxic to the HeLa cell line at the 150 μMconcentration for incubation during 12 hours. Cell death was confirmedby incubation with propidium iodide (PI), as shown in FIG. 18C. Asillustrated in the merged figures (FIGS. 18D and 18E),Yb³⁺[Zn(II)MC_(pyzHA)]and PI colocalize in the same cell compartment,and the signal was observed in the NIR region (FIG. 18B) and VIS region(FIG. 18C) originating from MC and PI, respectively.

The results shown in FIGS. 9A-9E are for the cells with the shortincubation and UV light exposure #1. While not shown, the NIR signalarising from Yb³⁺[Zn(II)MC_(pyzHA)] was obtained using epifluorescentimaging one month after the procedure for counter staining and fixingwas performed. The results after one month were compared with FIG. 9B(the NIR signal right after the staining and fixing method wasperformed), and the comparison illustrated that the stained and fixedcells maintained the same shape and morphology. With this experiment, anew photochemical phenomenon arising from the exposure of HeLa cancercells to UV-A light (377 nm) combined with the incubation of a highconcentration of Yb³⁺[Zn(II)MC_(pyzHA)]was observed. This effect wassimilar to cell fixation obtained classically with formaldehyde ormethanol. FIG. 9B also illustrates that Yb³⁺[Zn(II)MC_(pyzHA)]operatesas a NIR stain for the nucleus as well as for the cytoplasm of fixedHeLa cancer cells. The cell death was confirmed with the commerciallyavailable marker, propidium iodide (PI) (FIG. 9B). As illustrated in themerged figures (FIGS. 9D and 9E), Yb³⁺[Zn(II)MC_(pyzHA)]and PIcolocalize in the same cell compartments, and the signal was observed inthe NIR region (FIG. 9B) and VIS region (FIG. 9C) originating from MCand PI, respectively.

The results shown in FIGS. 7A-7E are for the cells with the shortincubation and no UV light exposure. In addition to the results in FIGS.18A-18E, the results in FIGS. 7A-7E demonstrate that the metallacrowns(e.g., Yb³⁺[Zn(II)MC_(pyzHA)]) are going exclusively in necrotic cells.The NIR staining of the nucleus and cytoplasm was also evident fromthese results.

The results shown in FIGS. 22A-22F are for the cells with the shortincubation and UV light exposure #2. These results also confirmed thatYb³⁺[Zn(II)MC_(pyzHA)]is going in necrotic cells. After incubation ofHeLa cells with Yb³⁺[Zn(II)MC_(pyzHA)]and propidium iodide for 20minutes, cells were illuminated with UV light for 5 minutes in order todamage cell membrane and induce cell death (necrosis and/or apoptosis).After 15 min of incubation, emission from propidium iodide was observedin the visible range, and after 5 more minutes, emission in NIR fromYb³⁺[Zn(II)MC_(pyzHA)]was observed. From this experiment, it can beconcluded that both dyes are specific for dead and apoptotic cells withdifferent kinetics.

The results shown in FIGS. 23A-23C are for the cells with the shortincubation and UV light exposure #3. The same photochemical effect seenwith the HeLa cell line occurred with the MSC cells. In the case of MSCcells, the Yb³⁺[Zn(II)MC_(pyzHA)]is also acting as an NIR dye and cellfixative.

Example 2 NIR Epifluorescent Microscopy of theYb³⁺[Zn(II)MC_(pyzHA)]Complex with HeLa Cancer Cells Treated withN-acetyl cysteine

Formation of extracellular vesicules is a result of cellular response onoxidative stress which indicates excessive production of ROS (Reactiveoxygen species). In order to investigate if ROS are responsible for dualfunction of Yb³⁺[Zn(II)MC_(pyzHA)], HeLa cancer cells were treated withNAC (N-acetyl cysteine), which is a well known antioxidant thatefficiently blocks production of reactive oxygen species (ROS).

Experimental Conditions

HeLa (Human Epithelial Ovarian Carcinoma) cell line obtained from ATCC(Molsheim, France) was cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS),1% of 100× non-essential aminoacid solution, 1% of L-glutamine(GlutaMAX) and 1% of streptomycin/penicillin antibiotics. Cells wereseeded in a 8-well Lab Tek Chamber coverglass (Nunc, Dutsher S.A.,Brumath, France) at a density of 6×10⁴ cells/well and cultured at 37° C.in 5% humidified CO₂ atmosphere.

NAC Incubation First: After 24 hours, the cell culture media wasremoved. Some of the cells were washed twice with OPTI-MEM® media (roomtemperature), incubated with N-acetyl cysteine (NAC) for 15 minutes,washed twice with OPTI-MEM® media and incubated with 150 μM ofYb³⁺[Zn(II)MC_(pyzHA)]for 15 minutes.

Yb³⁺[Zn(II)MC_(pyzHA)]Incubation First: After 24 hours, the cell culturemedia was removed. Some of the cells were washed twice with OPTI-MEM®media (room temperature), and incubated with 150 μM ofYb³⁺[Zn(II)MC_(pyzHA)]for 15 minutes. N-acetyl cysteine (NAC) was added,and the cells were incubated for another 30 minutes. The cells werewashed twice with OPTI-MEM® media.

Prior to epifluorescent imaging, the cells were washed trice withOPTI-MEM® (room temperature) in order to remove any non-specificallybound Yb³⁺[Zn(II)MC_(pyzHA)]complex. The cells were observed with aZeiss Axio Observer Z1 fluorescence inverted microscope (Zeiss, Le Pecq,France) equipped with an EMCCD Evolve 512 (Roper Scientific) photometriccamera or ORCA-R2 high resolution CCD camera. The light source, ZeissHXP 120, was combined with the following filter cubes: 377 nm band pass50 nm filter for the excitation and long pass filter 805 nm or 996 nmband pass filter 70 nm for Yb^(III) emission in the NIR range.

Experimental Results

The results from the NAC Incubation First experiment are shown in FIGS.25A through 25C. FIG. 25B shows a very good signal in NIR with veryshort exposure time (400 ms), which indicates that modulation ofmetabolism through signal transduction pathways in cancer cells has aninfluence on the efficiency of Yb³⁺[Zn(II)MC_(pyzHA)]complex as an NIRdye, as well as on their internalization in the cells. From theseresults, it can be concluded that production of ROS is not responsiblefor dual function of Yb³⁺[Zn(II)MC_(pyzHA)]complex.

The results from the Yb³⁺[Zn(II)MC_(pyzHA)] Incubation First experimentare shown in FIGS. 26A through 26C. FIG. 26B shows the NIR signal ofYb³⁺[Zn(II)MC_(pyzHA)]just in the cell culture media, outside of thecells. These results do not show internalization of the MC complexesinside of the cells. These results indicate that if the metabolism ofthe cells is not modified (in the specific way that it is stoppingprodution of ROS species), there will not be internalization ofYb³⁺[Zn(II)MC_(pyzHA)]in the cells. These results confirm the results ofthe previous experiment (NAC Incubation First) where it was shown thatmodulation of metabolism through signal transduction pathways in cancercells has an influence on the efficiency of Yb³⁺[Zn(II)MC_(pyzHA)] (alsodescribed as YbZn₁₆(pyz)₁₆MC) complex as an NIR dye, as well as on theirinternalization in the cells.

Example 3 Confocal Microscopy of the Yb³⁺[Zn(II)MC_(pyzHA)] Complex withHeLa Cancer Cells or Mesenchymal Stem Cells (MSC)

Experimental Conditions

For these confocal microscopy experiments, the HeLa or MSC cells wereprepared in the same way as for the epifluorescence microscopyexperiments (see Example 1, Short incubation, UV light exposure #1 and#3). The cells were observed with a confocal laser scanning microscopy(CLSM) on a Zeiss Axiovert 200M microscope equipped with LSM 510 Metascanning device(Zeiss, France). Complexes were excited with Argon laserat 458 nm and emission signal was collected from 499 nm to 799 nm.During this experiment, a 63× Plan-Apochromat objective was used (2×zoom).

Experimental Results

FIGS. 19A-19C are the confocal images for the HeLa cells and FIGS.24A-24C are the confocal images for the MSC stem cells. In FIGS. 19B and24B, emission was observed from Yb³⁺[Zn(II)MC_(pyzHA)], arising from theligands, in the visibile range. This emission enabled the use ofconfocal microscopy. This experiment confirmed thatYb³⁺[Zn(II)MC_(pyzHA)] (and other Ln³⁺[Zn(II)MC_(pyzHA)]) arespecifically located in nucleus as well as in cytoplasm of HeLa cancercells and of MSC stem cells.

Example 4 Cytotoxicity Assay

In order to determine if the LnZn₁₆(pyz)₁₆MC complexes(Ln³⁺[Zn(II)MC_(HA)]) are toxic for live cells, cytotoxicity tests withAlamar blue were performed.

Alamar blue is viability reagent which functions as a cell healthindicator by using the reducing power of living cells to quantitativelymeasure the proliferation of different human and animal cell lines. Thisenables the cytotoxicity of different chemical complexes to bedetermined. The active ingredient of Alamar Blue is resazurin, which isnon-toxic and cell permeable compound, and upon entering in the cells,it is reduced to highly fluorescent resorufin.

Experimental Conditions

The cytotoxicity tests were performed with Alamar blue assay(Invitrogen, France). HeLa cells were seeded in 96-well plate at thedensity of 1*10⁴ cells per well and cultured at 37° C. in 5% humidifiedCO₂ atmosphere. After 24 hours of attachment, the cells were incubatedwith different concentrations of Yb³⁺[Zn(II)MC_(pyzHA)] in cell culturemedium for 24 and 48 hours followed by incubation with Alamar blue (10%v/v) for 3 to 4 hours at 37° C. in 5% humidified CO₂ atmosphere.

Fluorescence of Alamar blue was measured with plate reader (Victor 3V,Perkin Elmer, France), with excitation at 530 nm and emission at 590 nm,for cells incubated with Yb³⁺[Zn(II)MC_(pyzHA)] and for untreated cells(control).

Experimental Results

The cytotoxicity test results for the cells exposed to differentconcentrations of Yb³⁺[Zn(II)MC_(pyzHA)] for 24 and 48 hours are shownin FIG. 13, and the cytotoxicity test results for the cells exposed todifferent concentrations of Yb³⁺[Zn(II)MC_(pyzHA)] for 5 hours are shownin FIG. 6. As illustrated in FIG. 6, Yb³⁺[Zn(II)MC_(pyzHA)]started to besignificantly toxic from 45 μM concentration, where approximately 70% ofcells remained alive.

Example 5 NIR Epifluorescent Microscopy of the Nd³⁺[Zn(II)MC_(pyzHA)]Complex with HeLa Cancer Cells

Experimental Conditions

HeLa cells were cultured and seeded as previously described inExample 1. In this example, Nd³⁺[Zn(II)MC_(pyzHA)]complexes were used.The HeLa cells were washed twice with OPTI-MEM® media (roomtemperature), incubated with a solution of 150 μMNd³⁺[Zn(II)MC_(pyzHA)]complex in OPTI-MEM® media (supplemented with 2%of FBS at 37° C. in 5% CO₂ atmosphere) for 15 minutes, illuminated withUV light (377 nm) for 8 minutes, and then were allowed to continueincubating for 1 hour. The continued incubation enabled theinternalization of the complex.

Prior to epifluorescent imaging, the HeLa cells were washed trice withOPTI-MEM® (room temperature) in order to remove any non-specificallybound Nd³⁺[Zn(II)MC_(pyzHA)] complex. The cells were observed with aZeiss Axio Observer Z1 fluorescence inverted microscope (Zeiss, Le Pecq,France) equipped with an EMCCD Evolve 512 (Roper Scientific) photometriccamera or ORCA-R2 high resolution CCD camera. The light source, ZeissHXP 120, was combined with the following filter cubes: 377 nm with 50 nmband pass filter for the excitation and long pass filter 805 nm for Nd³⁺emission in the NIR range.

Experimental Results

The epifluorescent imaging results are shown in FIGS. 24A-24C. The HeLacells were fixed and stained with the Nd³⁺[Zn(II)MC_(pyzHA)] complex,just as they were for the Yb³⁺[Zn(II)MC_(pyzHA)]complex.

Example 6 NIR Epifluorescent Microscopy of theNd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] Complex with HeLa Cancer Cells

Experimental Conditions

HeLa cells were cultured in the same manner as described in Example 5.After 24 hours of cell culturing in a 8-well Lab Tek Chamber coverglass(Nunc, Dutsher S. A., Brumath, France), cell culture media was removed,cells were washed twice with OPTI-MEM® media (room temperature).

The cells were incubated with a solution of 200 μMNd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] (or NdZn₁₆(pyz)_(x)(quino)_(y)MC)complex in OPTI-MEM® media (supplemented with 2% of FBS at 37° C. in 5%CO₂ atmosphere) for 3 hours. In order to induce cell apoptosis and toallow internalization of the Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] complexinside of the nucleus, the incubating cells were illuminated with UV-Alight for 10 minutes, followed by continued incubation for another 1.5hours. Some of the cells were also then incubated with 3 μM propidiumiodide for 5 minutes.

Prior to epifluorescent imaging, the cells were washed trice withOPTI-MEM® media (room temperature) in order to remove anynon-specifically bound Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] complex. Thecells were observed with a Zeiss Axio Observer Z1 fluorescence invertedmicroscope (Zeiss, Le Pecq, France) equipped with an EMCCD Evolve 512(Roper Scientific) photometric camera or ORCA-R2 high resolution CCDcamera. The light source, Zeiss HXP 120, was combined with the followingfilter cubes:

-   -   a. 377 nm with 50 nm band pass filter for the excitation and        long pass filter 805 nm or 895 nm with 90 nm band pass filter        for Nd^(III) emission in the NIR range.    -   b. 536 nm with 40 nm band pass for the excitation and long pass        filter 805 nm or 895 nm with 90 nm band pass filter for Nd^(III)        emission in the NIR range.    -   c. 480 nm with 50 nm band pass for the excitation and long pass        filter 805 nm or 895 nm with 90 nm band pass filter for Nd^(III)        emission in the NIR range.

Experimental Results

The results for the cells without UV exposure are shown in FIGS.16A-16C. MCs formed with two different ligands, PyzHA and QuinoHA,exhibit specific properties in biological conditions. These MCs serve asa NIR dye for the cell membrane (as evidenced in FIG. 16B).

Moreover, with this composition of MCs, the excitation wavelengthshifted toward lower energy, where these complexes can be excited up to536 nm. This shift in excitation energy is important because it allowpenetration through the tissue as well as better detection sensitivitybecause of lower absorption of the biological molecules (lipids,proteins, hemoglobin, etc.) for these wavelengths.

The results for the cells with UV exposure are shown in FIGS. 17A-17E.The exposure of HeLa cells to UV-A light (377 nm) for 10 minutes damagedthe cell membrane and induced apoptosis (programmed cell death) and/ornecrosis (unprogrammed cell death) of the cells. This allowed forinternalization of the Nd³⁺[Zn(II)MC_((pyzHA)x(quinoHA)y)] complex inthe nucleus (as illustrated in FIG. 17B). This experiment also indicatesthat the same photochemical effect (as seen with Yb³⁺[Zn(II)MC_(pyzHA)])takes place.

Example 7 Photostability/Photophysical Properties

Photostability Stress Tests

Photostability stress tests of Yb³⁺[Zn(II)MC_(pyzHA)] (Yb λ_(em)=980 nm)and Nd³⁺[Zn(II)MC_(pyzHA)] (Nd λ_(em)=1064 nm) and the commerciallyavailable dye for necrotic cells (Propidium Iodide) were performed. Thecomplexes (150 μM) or dye (50 μM) were dissolved in water and thecomplexes were also dissolved in OPTI-MEM® media. The MC solutions wereexposed to 5 hours of UV radiation (377 nm). The propidium iodidesolution was exposed to 5 hours of UV radiation (530 nm). The resultsare shown in FIGS. 18A and 18B. The results show that both MC complexeswere fully photostable during 5 hours of irradiation with 377 nm light,while the fluorescence intensity of the propidium iodide dropped half ofits initial intensity upon irradiation at 530 nm.

Photophysical properties of Yb^(3+]Zn(II)MC) _(pyzHA)]in the HeLa CancerCells

The Yb³⁺[Zn(II)MC_(pyzHA)] complexes were dissolved in OPTI-MEM® mediumwith 2% SVF (without cells) and were incorporated in HeLa cells, bothwith and without exposure to UV light. The results in are shown in FIG.20A (corrected and normalized excitation) and 20B (corrected andnormalized emission). These experiments showed that the spectroscopicproperties of MCs (Yb³⁺[Zn(II)MC_(pyzHA)]) can be controlled in cellculture media and in HeLa cancer cells under different experimentalconditions. The present inventors developed instrumentation in order tocollect photophysical properties of MCs inside of cancer cells. Theymeasured quantitative photophysical parameters (quantum yields and lifetimes) inside of the cells with equipment which is usually adapted formeasuring of (quantum yields and life times) in solids or inconcentrated solutions. The results showed that luminescent propertiesof the MC complexes did not change in the cells, as an indication thattheir structures remain intact.

The Yb³⁺[Zn(II)MC_(pyzHA)]complexes were collected together with cellsupernatant (SN) and with HeLa cells treated with N-acetyl cysteine,both with and without exposure to UV light. For SN1 and cells1,incubation with NAC took place for 30 minues, then the cells werewashed, and incubation with Yb³⁺[Zn(II)MC_(pyzHA)]took place for 15minutes. For SN2 and cells2, incubation with Yb³⁺[Zn(II)MC_(pyzHA)] tookplace for 15 minutes, and then NAC was added and incubation with NACtook place for 30 minutes. The results in are shown in FIGS. 20C(corrected and normalized excitation) and 20D (corrected and normalizedemission). The characteristic emission (FIG. 20B), arising from Yb f-ftransitions, show that luminescent properties of theYb³⁺[Zn(II)MC_(pyzHA)]complexes did not change in cell supernatant orinside of the HeLa cells (with/without exposure to UV light). Theseresults indicate that the cell structures remain intact.

Table 1, below, illustrates some of the photophysical parameters ofYb³⁺[Zn(II)MC_(pyzHA)]complexes in OPTI-MEM® media, cell supernatant(SN) and in HeLa cells (with/without exposure to UV light andwith/without treatment with N-acetyl cysteine).

TABLE 1 Quantum Condition yield/% τ₁/μs τ₂/μs OptiMEM 1.25(1) · 10⁻²6.45(5): 89%   24(1): 11% OptiMEM-UV 2.05(5) · 10⁻² 7.03(3): 61%21.5(1): 39% 1 h 30 min No UV: After 5 h of 1.52(3) · 10⁻² 6.47(4): 75%21.6(8): 25% incubation with HeLa cells without illumination UV: After 5h of 1.64(5) · 10⁻² 6.51(8): 65%   21(1): 35% incubation with HeLa cellswith UV illumination (during 8 min) In HeLa cells 2.14(6) · 10⁻² 23.6(7): 100% No UV NAC (SN1*) 1.08(1) · 10⁻² 7.27(2): 91%  50(2): 9%No UV NAC (in cells1*) n.a. 7.5(4): 6% 22.9(1): 94% No UV NAC (SN2*)1.00(4) · 10⁻² 6.72(3): 88% 28.4(1): 12% No UV NAC (in cells2*) n.a.7.3(1): 7% 23.6(2): 93% UV NAC (SN2*) 1.06(5) · 10⁻² 6.80(9): 75%  23(1): 25% UV NAC (in cells2*) n.a. 8.7(5): 9% 23.2(1): 91%

Yb³⁺[Zn(II)MC_(pyzHA)]exhibit promising photophysical properties, whichare among the highest values for quantum yields and luminescencelifetimes. The data in Table 1 are additional quantitative data showingthat the MC stays intact in cell culture media and in the cells underdifferent experimental conditions.

In cell culture media, the start of two lifetimes was observed, wherethe first lifetime is comparable with the one obtained forYb³⁺[Zn(II)MC_(pyzHA)] in H₂O while second lifetime was much longer.With the OPTI-MEM® media with Yb³⁺[Zn(II)MC_(pyzHA)] exposed to UVlight, a higher percentage of 2. lifetime (39%) was seen, while withoutexposure to UV, 2. lifetime was 11%.

From these results, it is very important to highlight that “in cells”was dominating longer component under all experimental conditions.

More experiments need to be performed in order to explain the fact thatinside of HeLa cells, the Yb³⁺[Zn(II)MC_(pyzHA)]had a dominating longlifetime, more than 90% for all experimental conditions.

Photophysical Properties of Nd³⁺[Zn(H)MC_(pyzHA)] in the HeLa CancerCells

The Nd³⁺[Zn(II)MC_(pyzHA)] complexes were incorporated into OPTI-MEM®medium (without cells), and were incorporated in HeLa cells, both withand without exposure to UV light. The results are shown in FIGS. 20E(corrected and normalized excitation) and 20F (corrected and normalizedemission). The characteristic emission (FIG. 21B), arising from Nd, showthat luminescent properties of the Nd³⁺[Zn(II)MC_(pyzHA)]complexes didnot change in OPTI-MEM® media or inside of the HeLa cells (with/withoutexposure to UV light). These results indicate that the cell structuresremain intact.

Table 2, below, illustrates some of the photophysical parameters of

Nd³⁺[Zn(II)MC_(pyzHA)]complexes in OPTI-MEM® media and in HeLa cells(with/without exposure to UV light).

TABLE 2 Compound Condition Quantum yield/% τ₁/μs τ₂/μs Nd^(III) MCOptiMEM 7.33(1) · 10⁻³ 0.256(6): 90%  1.1(1): 10% OptiMEM-UV 1 h 30 min 7.7(1) · 10⁻³ 0.352(2): 67% 0.874(7): 33%  No UV: After 5 h incubationwith HeLa cells 7.50(5) · 10⁻³ 0.261(1): 85% 0.98(1): 15% withoutillumination UV: After 5 h incubation with HeLa cells with UV  6.2(1) ·10⁻³ 0.376(3): 60% 0.83(2): 40% illumination (during 8 min) In HeLacells 0.808(8): 100%

The photophysical results for Nd³⁺[Zn(II)MC_(pyzHA)]in OPTI-MEM® mediaand in HeLa cells were similar to those for Yb³⁺[Zn(II)MC_(pyzHA)]. InOPTI-MEM® media, there started to be a longer component. When exposingthat medium to UV light, there was a higher contribution of the longercomponent. For Nd³⁺[Zn(II)MC_(pyzHA)]inside of HeLa cells, there was100% of the longer component.

Example 8 Raman Spectroscopy

Raman spectroscopy maps were collected for living HeLa cells, HeLa cellsthat were fixed with paraformaldehyde (PFA), HeLa cells that were fixedwith methanol (MeOH), and HeLa cells that were stained and fixed withYb³⁺[Zn(II)MC_(pyzHA)] using the example method disclosed herein (asdescribed in Example 1).

Fixation of the cells with PFA (paraformaldehyde): Cells were seeded inan 8-well Lab Tek Chamber coverglass, as previously described inExample 1. Cells were washed trice with OPTI-MEM® (room temperature) andincubated with 4% PFA solution in PBS (Phosphate Buffered Saline) for 30minutes at room temperature. After incubation with PFA, cells werewashed trice with OPTI-MEM® (room temperature) and new reduced mediumwas added on the cells (OPTI-MEM®+2% of SVF).

Fixation of cells with methanol: Cells were seeded in an 8-well Lab TekChamber coverglass, as previously described in Example 1. Cells werewashed trice with OPTI-MEM® (room temperature) and incubated withice-cold 100% methanol at −20° C. for 10 minutes. Cells were washedtrice with OPTI-MEM® (room temperature) and new reduced medium was addedon the cells (OPTI-MEM®+2% of SVF).

The areas corresponding to the cytoplasm and to the nucleus wereselected manually on the different maps, FIGS. 21A to 21D.

To study the changes between the different samples, a backgroundsubtraction was applied and the signal was collected in the range of 0-4000 cm⁻¹ in different cellular structures, cytoplasm and nucleus,allowing to observe the variations of specific vibrations arising fromthe nucleic acids and proteins, in particular, the CH vibrational bandsat 2800-3300 cm⁻¹ that reflect a distribution of proteins, lipids andcarbohydrates in cells and often used to localize cellular organelles,as well as OH band in the range of 3100-3650 cm⁻¹. The average spectrumcorresponding to the one with the maximum CH bands signal (e.g.cytoplasm and nucleus) was extracted for each sample. Results indicatevery similar biomolecular profiles of HeLa cells fixed withYb³⁺[Zn(II)MC_(pyzHA)] to the ones fixed with PFA and methanol. Thus,the main difference in respect to the living cells was the decrease ofthe 752 cm⁻¹ peak intensity corresponding to the cytochrome c which hasbeen already reported for the fixation with PFA (Okada et al. Proc NatlAcad Sci USA, 109 (2012) 28-32).

However, a more detailed analysis of Raman signal is required to fullyquantify and identify corresponding peaks in order to completelyunderstand changes in the biomolecular profile observed for the fixationwith Yb³⁺[Zn(II)MC_(pyzHA)].

Photobleaching experiments on epifluorescence microscope. During thephotobleaching experiments conducted under the epifluorescencemicroscope, necrotic HeLa cells labelled with PI were illuminated during8 min with 550 nm band pass 25 nm filter and images were captured every10 s with the following filter cube: 550 nm band pass 25 nm for theexcitation and 605 nm band pass 70 nm for the emission. The sameexperiment was performed with Yb³⁺[Zn(II)MC_(pyzHA)] which was excitedusing a 447 nm band pass 60 nm filter and images were captured every 10s with the following cube: 447 nm band pass 60 nm for the excitation andlong pass 805 nm filter for Yb³⁺ emission in the NIR range.

It is to be understood that the ranges provided herein include thestated range and any value or sub-range within the stated range. Forexample, a range from about 45 μM to about 400 μM should be interpretedto include not only the explicitly recited limits of about 90 μM toabout 400 μM, but also to include individual values, such as 99.5 μM,200 μM, etc., and sub-ranges, such as from about 100 μM to about 350 μM,etc. Furthermore, when “about” is utilized to describe a value, this ismeant to encompass minor variations (up to +/−10%) from the statedvalue.

Reference throughout the specification to “one example”, “anotherexample”, “an example”, and so forth, means that a particular element(e.g., feature, structure, and/or characteristic) described inconnection with the example is included in at least one exampledescribed herein, and may or may not be present in other examples. Inaddition, it is to be understood that the described elements for anyexample may be combined in any suitable manner in the various examplesunless the context clearly dictates otherwise.

In describing and claiming the examples disclosed herein, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise.

While several examples have been described in detail, it is to beunderstood that the disclosed examples may be modified. Therefore, theforegoing description is to be considered non-limiting.

1. A method for simultaneously fixing and staining cells, the methodcomprising: initially incubating the cells in a solution including aLn(III)Zn₁₆(HA ligand)₁₆ metallacrown complex, wherein the HA ligand isa hydroximate ligand; exposing the incubating cells to ultraviolet (UV)light; and continuing to incubate the cells in the solution after UVlight exposure.
 2. The method of claim 1 wherein: the solution includesthe Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex in a medium; and aconcentration of the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex inthe solution ranges from about 90 μM to about 400 μM.
 3. The method ofclaim 1, further comprising culturing the cells prior to incubating thecells in the solution.
 4. The method of claim 1, wherein: initiallyincubating the cells is accomplished for a time (T1) ranging from about10 minutes to about 3 hours; exposing the incubating cells to UV lightis accomplished for a time (T2) ranging from about 5 minutes to about 10minutes; and continuing to incubate the cells is accomplished for a time(T3) ranging from about 1 hour to about 2 hours.
 5. The method asdefined in claim 4, further comprising exposing the incubating cells toadditional UV light for a time (T4) ranging from about 1 minute to about5 minutes.
 6. The method of claim 1, wherein the HA ligand of theLn(III)Zn₁₆(HA ligand)₁₆ metallacrown complex is selected from the groupconsisting of pyrazinehydroximate, quinoxalinehydroximate,quinaldinehydroximate, and combinations thereof.
 7. The method asdefined in claim 6 wherein: the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrowncomplex is one of: [Ln(III)Zn₁₆(pyrazinehydroximate)₁₆(pyridine)₈]counter ion; [Ln(III)Zn₁₆(quinoxalinehydroximate)₁₆(pyridine)₈] counterion; or [Ln(III)Zn₁₆(quinaldinehydroximate)₁₆(pyridine)₈] counter ion;wherein the Ln(M) is selected from the group consisting of Y³⁺, La³⁺,Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺,Yb³⁺, and Lu³⁺; and wherein the counter ion is selected from the groupconsisting of a triflate, a mesylate, a besylate, a camsylate, anedisylate, an estolate, an esylate, a napsylate, a tosylate, a fluoride,a chloride, a bromide, an iodide, a nitrate, a sulfate, a carbonate, anacetate, a phosphate, and a sulfonate.
 8. The method as defined in claim6 wherein: the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex includes amixture of metallacrown complexes; each species in the metallacrownmixture is [Ln(III)Zn₁₆(pyrazinehydroximate)_(x)(quinoxalinehydroximate)_(y)(pyridine)₈]counter ion, wherein x+y=16; wherein the Ln(III) is selected from thegroup consisting of Y³⁺, La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Pm³⁺, Sm³⁺, Eu³⁺, Gd³⁺,Tb³⁺, Dy³⁺, Ho³⁺, Er³⁺, Tm³⁺, Yb³⁺, and Lu³⁺; and wherein the counterion is selected from the group consisting of a triflate, a mesylate, abesylate, a camsylate, an edisylate, an estolate, an esylate, anapsylate, a tosylate, a fluoride, a chloride, a bromide, an iodide, anitrate, a sulfate, a carbonate, an acetate, a phosphate, and asulfonate.
 9. The method of claim 1, wherein the medium is aserum-supplemented medium.
 10. The method of claim 1, wherein the UVlight is UV-A light.
 11. The method of claim 1, wherein: UV lightexposure induces death of at least some of the cells; and aftercontinuing to incubate the cells, the Ln(III)Zn₁₆(HA ligand)₁₆metallacrown complex is located in nuclei and cytoplasm of least somedead cells.
 12. An optical imaging method, comprising: formingsimultaneously fixed and stained cells by: initially incubating cells ina solution including a Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex,wherein the HA ligand is a hydroximate ligand; exposing the incubatingcells to ultraviolet (UV) light; and continuing to incubate the cells inthe solution after UV light exposure; and exposing the simultaneouslyfixed and stained cells to an optical imaging technique selected fromthe group consisting of epifluorescence microscopy, confocal microscopy,and combinations thereof.
 13. The optical imaging method as defined inclaim 12, further comprising any of: tuning an excitation response ofthe simultaneously fixed and stained cells by changing the Ln(III) ofthe Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex; or tuning an emissionresponse of the simultaneously fixed and stained cells by changing thehydroximate ligand of the Ln(III)Zn₁₆(HA ligand)₁₆ metallacrown complex.14. A method for selective labelling of necrotic cells, the methodcomprising incubating the cells in a solution including a Ln(III)Zn₁₆(HAligand)₁₆ metallacrown complex, wherein the HA ligand is a hydroximateligand.
 15. The method of claim 8, wherein x ranges from 8 to 13 and yranges from 3 to 8.